TWI731602B - Matrix metalloproteinase-1 monoclonal antibody, detection kit and detection method thereof - Google Patents

Abstract

A MMP-1 monoclonal antibody is provided in the disclosure. A variable region of a heavy chain of the monoclonal antibody has an amino acid sequence including i) CDR1 selected from a group consisting of SEQ ID NO: 1, 7 and 13, ii) CDR2 selected from a group consisting of SEQ ID NO:2, 8 and 14, and iii) CDR3 selected from a group consisting of SEQ ID NO:3, 9 and 15. A variable region of a light chain of the monoclonal antibody has an amino acid sequence including i) CDR1 selected from a group consisting of SEQ ID NO:4, 10 and 16, ii) CDR2 selected from a group consisting of SEQ ID NO:5, 11 and 17, and iii) CDR3 selected from a group consisting of SEQ ID NO:6, 12 and 18. Polynucleotides encoding the monoclonal antibody and polynucleotides complement to the above-mentioned polynucleotides are also provided. MMP-1 detection kit containing the above-mentioned monoclonal antibody and MMP-1 detection method are further provided in the disclosure.

Description

Monoclonal antibody of type I matrix metalloproteinase, its detection kit and its detection method law

The present disclosure relates to detection methods for MMP-1 and oral cancer, and particularly relates to MMP-1 monoclonal antibodies, detection kits containing MMP-1 monoclonal antibodies, and detection methods using the detection kits.

According to statistics from the World Health Organization, there are more than 529,000 new cases of oral cancer each year. As the incidence is increasing year by year, it is expected that the number will reach 856,000 a year by 2035. In Taiwan, according to the 2016 death cause statistics of the National Health Administration of the Ministry of Health and Welfare (hereinafter referred to as the National Health Administration), oral cancer, oropharyngeal cancer and hypopharyngeal cancer accounted for the fourth leading cause of death among all male malignant tumors. Every year, the number of newly diagnosed patients is about 7,000, and the death toll is about 3,000. Between 2012 and 2016, the survival rates of oral cancer patients from stage I to stage IV were 79.9%, 71.0%, 56.5% and 35.6% respectively. If detected early, the survival rate will be improved.

At present, oral mucosal screening is an important method for clinical evaluation of oral cancer, and the results of tissue biopsy at the lesion are used as the diagnosis basis. However, the current mouth The screening of the cavum mucosa must be performed by professional oral health personnel (such as: dentist, oral hygienist, dental therapist, oral hygienist, etc.). In many areas, the diagnosis of oral cancer will be delayed due to the lack of professional oral health personnel. Out of time. Therefore, if the samples can be analyzed for biomarkers, the early diagnosis rate of oral cancer can be improved.

At present, oral cancer is not a tumor marker that is routinely identified. According to a report in the 2016 Proceedings of the National Academy of Sciences of the United States of America (PNAS), the mass spectrometry method was used to quantify type 1 matrix metalloproteinase (MMP-1), It showed that the expression level of MMP-1 between the saliva samples of the patient and the control group was as high as 83 times (the fold difference of other molecules was -1.3 to 5.5 times), and it was the molecule with the most potential as a tumor marker of oral cancer.

Since the current mass spectrometry quantitative analysis method for MMP-1 is not suitable for routine quantitative analysis of a large number of samples, it is hoped to develop a detection kit for MMP-1 for the screening of oral cancer.

In some embodiments of the present disclosure, a monoclonal antibody is provided, which includes a heavy chain variable region sequence and a light chain variable region sequence. The heavy chain variable region sequence comprises i) CDR1 selected from the group consisting of SEQ ID NO: 1, 7 and 13, ii) CDR2 selected from the group consisting of SEQ ID NO: 2, 8 and 14, and iii) selected from SEQ ID NO: 3, 9 and CDR3 of the group consisting of 15; the light chain variable region sequence comprises i) CDR1 selected from the group consisting of SEQ ID NO: 4, 10 and 16, ii) selected from the group consisting of SEQ ID NO: 5, 11 and The CDR2 of the group consisting of 17 and iii) the CDR3 of the group consisting of SEQ ID NO: 6, 12 and 18.

In some embodiments, the heavy chain variable region sequence includes the amino acid sequence of SEQ ID NO: 1, 2, and 3, and the light chain variable region sequence includes the amino acid sequence of SEQ ID NO: 4, 5, and 6. Acid sequence.

In some embodiments, the heavy chain variable region sequence includes the amino acid sequence of SEQ ID NO: 7, 8, and 9, and the light chain variable region sequence includes the amino acid sequence of SEQ ID NO: 10, 11, and 12. Acid sequence.

In some embodiments, the heavy chain variable region sequence includes the amino acid sequences of SEQ ID NOs: 13, 14, and 15, and the light chain variable region sequence includes the amino acid sequences of SEQ ID NOs: 16, 17, and 18. Acid sequence.

In some embodiments of the present disclosure, a polynucleotide is provided, which encodes the aforementioned amino acid sequence, or has a sequence complementary to the nucleotide sequence encoding the aforementioned monoclonal antibody.

In some embodiments of the present disclosure, a detection kit is provided. The detection kit includes a monoclonal antibody A, wherein the monoclonal antibody A includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 1, 2, and 3; the light chain variable region includes the amino acid sequence of SEQ ID NO: 4, 5, and 6.

In some embodiments, the detection kit further includes an enzyme immunoassay reagent kit, a colloidal gold immunoassay strip, or a combination thereof.

In some embodiments, the enzyme immunoassay reagent kit includes monoclonal antibody B. Monoclonal antibody B includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the amino acid sequences of SEQ ID NO: 7, 8, and 9; the light chain variable region includes SEQ ID NO: 10 , 11, and 12 amino acid sequences.

In some embodiments, the monoclonal antibody B is linked to a chromogenic group.

In some embodiments, the colloidal gold immunization test strip contains monoclonal antibody C. Monoclonal antibody C includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the amino acid sequences of SEQ ID NOs: 13, 14, and 15; the light chain variable region includes SEQ ID NO: 16 , 17, and 18 amino acid sequences.

In some embodiments, the colloidal gold immunization test strip further comprises monoclonal antibody A, and the monoclonal antibody A in the colloidal gold immunization test strip is connected to gold particles.

In some embodiments of the present disclosure, an in vitro detection method for detecting type 1 matrix metalloproteinases is provided, which comprises using the aforementioned detection kit to detect type 1 matrix metalloproteinases in a sample.

In some embodiments, the sample contains body fluid or blood.

In some embodiments, the body fluid comprises oral secretions or respiratory secretions.

1: Colloidal gold immune test piece

110: plastic gold pad

120: measurement area

121: Internal Control Line

122: Measuring Line

In order to make the above and other objectives, features, advantages and embodiments of the present invention more comprehensible, the description of the accompanying drawings is as follows:

Figures 1A and 1B illustrate the use of enzyme immunoassay in an embodiment of the present invention Method to determine the binding ability of self-produced monoclonal antibodies to type I matrix metalloproteinase;

Figures 2A and 2B illustrate that in an embodiment of the present invention, four better-matched monoclonal antibodies are used as capture antibodies and detection antibodies for enzyme immunoassays, targeting normal people (Figure 2A) and oral cancer patients. (Figure 2B) Concentration distribution graph of saliva measured by enzyme immunoassay method;

Figure 3A illustrates a schematic diagram of a colloidal gold immunoassay strip in an embodiment of the present invention;

Figure 3B illustrates a schematic diagram showing the level of detection of colloidal gold immunoassay strips with a colorimetric card in an embodiment of the present invention; and

Fig. 4 illustrates the cross-comparison diagram of the colloidal gold immunoassay test strip and the enzyme immunoassay reagent group in an embodiment of the present invention.

In order to make the description of the present invention more detailed and complete, the following describes the embodiments and specific examples of the present invention in detail; but this is not the only way to implement or use the specific embodiments of the present invention. The embodiments disclosed below can be combined or substituted with each other under beneficial circumstances, and other embodiments can also be added to an embodiment without further description or description. In the following description, many specific details will be described in detail to enable the reader to fully understand the following embodiments. However, the embodiments of the present invention may be practiced without these specific details.

In this article, unless there are special restrictions on the article in the text, "一" and "the" can generally refer to one or more. Will understand further Yes, "include", "include", "have" and similar words used in this text indicate the recorded features, regions, integers, steps, operations, elements and/or components, but do not exclude other features , Area, integer, step, operation, element, component, and/or group thereof.

Although a series of operations or steps are used in the following to illustrate the method disclosed herein, the sequence of these operations or steps should not be construed as a limitation of the present invention. For example, certain operations or steps may be performed in a different order and/or simultaneously with other steps. In addition, not all operations, steps, and/or features must be performed to implement the embodiments of the present invention. Furthermore, each operation or step described herein may include several sub-steps or actions.

In this article, "CDR" (complementarity determining regions) refers to the area where antigen and antibody contact, and is part of the variable region of an antibody. Generally, there are three CDRs in the variable region of an antibody, namely CDR1 and CDR2. , And CDR3.

As used herein, "derived sequence" refers to a sequence modified at the 3'end or 5'end of the nucleotide sequence.

In some embodiments of the present disclosure, a monoclonal antibody that can recognize type 1 matrix metalloproteinase (MMP-1) is proposed, which includes a heavy chain variable region sequence and a light chain variable region sequence. The heavy chain variable region sequence comprises i) CDR1 selected from the group consisting of SEQ ID NO: 1, 7 and 13, ii) CDR2 selected from the group consisting of SEQ ID NO: 2, 8 and 14, and iii) CDR3 selected from the group consisting of SEQ ID NO: 3, 9 and 15; the light chain variable region sequence comprises i) selected from SEQ ID NO: 4, CDR1 of the amino acid sequence of the group consisting of 10 and 16, ii) CDR2 selected from the group consisting of SEQ ID NO: 5, 11 and 17, and iii) selected from the group consisting of SEQ ID NO: 6, 12 and CDR3 of the group consisting of 18.

In some embodiments, the heavy chain variable region sequence includes the amino acid sequence of SEQ ID NO: 1, 2, and 3, and the light chain variable region sequence includes the amino acid sequence of SEQ ID NO: 4, 5, and 6. Acid sequence. In some embodiments, the heavy chain variable region sequence includes the amino acid sequence of SEQ ID NO: 7, 8, and 9, and the light chain variable region sequence includes the amino acid sequence of SEQ ID NO: 10, 11, and 12. Acid sequence. In some embodiments, the heavy chain variable region sequence includes the amino acid sequences of SEQ ID NOs: 13, 14, and 15, and the light chain variable region sequence includes the amino acid sequences of SEQ ID NOs: 16, 17, and 18. Acid sequence.

In some embodiments of the present disclosure, a polynucleotide is provided, which can encode the aforementioned amino acid sequence, or has a sequence complementary to the aforementioned polynucleotide sequence. In one embodiment, the polynucleotide may further include a derivative sequence.

In some embodiments of the present disclosure, a test kit is provided, wherein the test kit can be used to test clinical samples, such as body fluids (for example, oral secretions or respiratory secretions) or blood. The test kit contains monoclonal antibody A. The variable region of the heavy chain of monoclonal antibody A includes the amino acid sequences of SEQ ID NOs: 1, 2, and 3; the variable region of the light chain of monoclonal antibody A includes the amines of SEQ ID NO: 4, 5, and 6. Base acid sequence. In some embodiments, the heavy chain variable region of monoclonal antibody A comprises CDR1 as SEQ ID NO: 1, CDR2 as SEQ ID NO: 2, and CDR3 as SEQ ID NO: 3. The light chain variable region of monoclonal antibody A includes the amino acid sequence of CDR1 as SEQ ID NO: 4, CDR2 as SEQ ID NO: 5, and CDR3 as SEQ ID NO: 6.

In some embodiments, the detection kit includes an enzyme immunoassay reagent kit, a colloidal gold immunoassay strip, or a combination thereof.

In some embodiments, the enzyme immunoassay reagent kit adopts sandwich enzyme-linked immunosorbent assay (sandwich ELISA), and uses monoclonal antibody A as a capture antibody. In one embodiment, the enzyme immunoassay reagent kit includes monoclonal antibody A and monoclonal antibody B, wherein monoclonal antibody A is used as a capture antibody, and monoclonal antibody B is connected to a chromogenic group to serve as a detection antibody (detection antibody) . The variable region of the heavy chain of monoclonal antibody B includes the amino acid sequences of SEQ ID NOs: 7, 8, and 9, and the variable region of the light chain of monoclonal antibody B includes the amines of SEQ ID NOs: 10, 11, and 12. Base acid sequence. In one embodiment, the heavy chain variable region of monoclonal antibody B comprises the amino acid sequence of CDR1 as SEQ ID NO: 7, CDR2 as SEQ ID NO: 8, and CDR3 as SEQ ID NO: 9; monoclonal antibody The light chain variable region of B includes the amino acid sequence of CDR1 as SEQ ID NO: 10, CDR2 as SEQ ID NO: 11, and CDR3 as SEQ ID NO: 12. In one embodiment, the chromogenic group includes a fluorescent group or a chemiluminescent group (for example, horseradish peroxidase (HRP)).

It is worth noting that in some embodiments of the present disclosure, it is disclosed that human recombinant MMP-1 (full-length MMP-1) is used as an immunogen, since The line produced 11 monoclonal antibodies, and screened out the monoclonal antibody pairs that can be used in the enzyme immunoassay reagent group (capture antibody: monoclonal antibody A; and detection antibody: monoclonal antibody B), which overcomes the monoclonal antibody The ability to bind MMP-1 that may be derived from linking chromophore groups is greatly reduced, the background value that often occurs in the detection of clinical samples is too high, and the sensitivity is insufficient. Compared with other pairs (some pairs cannot even detect MMP-1), it has Better sensitivity. In addition, it needs to be emphasized that such pairing has no correlation with the ability of monoclonal antibodies to bind to MMP-1. It is worth mentioning that, compared with the capture antibody, it uses commercially available antibodies (the immunogen is the 20th-469th amino acid of MMP-1), and uses MMP-1 without protease activity as the immunogen. Antibodies, or antibody pairings of multiple antibodies produced by immunization with MMP-1 fragments (such as any fragment of the 100th-300th amino acid of MMP-1, or fragments without protease activity) as immunogens, The monoclonal antibody pairing of some embodiments of the present disclosure has higher sensitivity.

In some embodiments, the colloidal gold immune test strip comprises a glue gold pad and a measurement plate, which overlap each other. The glue gold pad is sprayed with a glue gold pad solution containing a detection antibody-gold particle coupling, and the surface of the measurement plate has nitrocellulose. The membrane, on which the solution containing the capture antibody is applied, serves as a measurement line. In one embodiment, the colloidal gold immunization test strip contains monoclonal antibody A and monoclonal antibody C. Monoclonal antibody A is used as a detection antibody connected to gold particles, and monoclonal antibody C is used as a capture antibody. The heavy chain variable region of monoclonal antibody C includes the amino acid sequences of SEQ ID NOs: 13, 14, and 15, and the light chain variable region of monoclonal antibody C includes the amines of SEQ ID NOs: 16, 17, and 18. Base acid sequence. In some embodiments, the heavy chain variable region of monoclonal antibody C comprises the amino acid sequence of CDR1 as SEQ ID NO: 13, CDR2 as SEQ ID NO: 14, and CDR3 as SEQ ID NO: 15; monoclonal antibody The light chain variable region of C includes the amino acid sequence of CDR1 as SEQ ID NO: 16, CDR2 as SEQ ID NO: 17, and CDR3 as SEQ ID NO: 18.

It is worth noting that in some embodiments of the present disclosure, from the monoclonal antibody pairs that can be used in the enzyme immunoassay reagent group, a combination of monoclonal antibodies that can be used in the colloidal gold immunoassay (capture antibody: monoclonal antibody C; and detection antibody: monoclonal antibody A). The detection antibody overcomes the problem that some monoclonal antibodies cannot be connected to gold particles, smoothly connects to gold particles, and has better sensitivity than other combinations. In addition, it also revealed that the pair with the best sensitivity in the enzyme immunoassay reagent group may not show the best sensitivity in the colloidal gold immunoassay test piece. That is, if one intends to apply the antibody pairing in one detection system to other detection systems, the quality of sensitivity still needs to be verified by actual experiments, and it is not necessarily positively correlated with existing known detection systems.

In some embodiments of the present disclosure, a method for detecting type 1 matrix metalloproteinases is provided, which includes using the aforementioned detection kit to detect type 1 matrix metalloproteinases in a sample, for example, by interpreting the enzymes in the detection kit The immunoassay reagent set, the colloidal gold immunoassay test piece, or the aforementioned combination of the presence or absence of color or the quantitative reading of the color (such as optical density (Optical Density value; OD value), detection of the presence or absence of type 1 matrix metalloproteinases) Content. In some embodiments, the sample contains body fluids (such as oral secretions or respiratory secretions) or blood. In the implementation method, the detection kit can use the enzyme immunoassay reagent kit for quantification, and use the colloidal gold immunoassay test piece for quick screening, so that qualitative and quantitative results can be obtained at the same time.

Since MMP-1 can be used as a detection index for oral cancer, the MMP-1 detection kits and detection methods provided by some embodiments of the present disclosure can be simultaneously used for oral cancer screening. Such an application does not need to be performed by professional oral health personnel, which can increase the popularity of screening, accelerate the diagnosis of oral cancer patients, and increase the probability of cure.

In order to further illustrate the monoclonal antibodies, polynucleotides, detection kits, and detection methods of type 1 matrix metalloproteinases provided by various embodiments of the present disclosure, the following implementations are carried out. It should be noted that the following embodiments are provided for exemplary purposes only, rather than limiting the present invention.

Example 1: The development process of monoclonal antibodies

First, using recombinant human MMP-1 protein that retains protease activity as an immunogen, through the immune response of mice, a mouse MMP-1 monoclonal antibody cell line was developed, and 11 mouse monoclonal antibody cells were screened out. Strains, collected the cell sap and purified each monoclonal antibody, and named them as: 1-8A12 strain, 4-26 strain, 6-2 strain, 20-4 strain, 30-22 strain, 31-34 strain, 44- 28 strains, 57-41 strains, 61-24 strains, 73-1 strains and 79-4 strains.

Example II. 1. Enzyme immunoassay reagent set for detecting MMP-1-test the ability of each monoclonal antibody to bind to MMP-1

In order to test whether each monoclonal antibody can specifically bind to MMP-1, direct enzyme immunoassay was used after serial dilution of the aforementioned 11 monoclonal antibodies The direct ELISA was used to test the binding reaction of monoclonal antibodies to human recombinant MMP-1, and the results are shown in Figure 1A.

Figure 1A shows that each monoclonal antibody will specifically bind to MMP-1, and different monoclonal antibodies have different binding abilities to MMP-1. The binding ability from strong to weak is in order: 6-2 strain>73 -1 strain>1-8A12 strain>4-26 strain>20-4 strain>31-34 strain>44-28 strain>61-24 strain>30-22 strain>57-41 strain>79-7 strain.

In addition, to test whether the monoclonal antibody can be used as a detection antibody in the enzyme immunoassay, that is, whether the monoclonal antibody still has the binding ability of MMP-1 after attaching the chromogenic group. After each monoclonal antibody is connected to HRP, the binding ability test of MMP-1 is carried out with the flow in Figure 1A above, and the results are shown in Figure 1B.

Figure 1B shows that each monoclonal antibody still has the ability to specifically bind to human recombinant MMP-1 after being connected to HRP. However, compared to Figure 1A, the monoclonal antibody linked to HRP showed a weaker binding ability to human recombinant MMP-1, from strong to weak: 73-1-HRP>4-26- HRP=6-2-HRP>44-28-HRP>31-34-HRP>1-8A12-HRP>20-4-HRP>61-24-HRP>57-41-HRP>30-22-HRP> 79-7-HRP.

Example 2: 2. Enzyme immunoassay reagent set for detecting MMP-1-screening antibody pairing

The above 11 monoclonal antibodies were used as the capture antibody and the detection antibody (connected to HRP) in the enzyme immunoassay reagent group, and the pairwise test was performed. Although in theory, 11x10=110 combinations of pairs will be generated, but However, because 3 strains of monoclonal antibodies performed extremely poorly in the pairing test, they were eliminated. In fact, a total of 92 combinations of pairing tests were completed. Then, a better pairing that can detect MMP-1 was screened out.

The test process of each pairing, taking the 1-8A12 strain as an example, is explained as follows: 1. Use the 1-8A12 strain as the capture antibody and 4-26-HRP as the detection antibody to perform Sandwich ELISA. The capture antibody contains 4 concentrations, and the detection antibody has 6 concentrations. A set of pairing tests a total of 24 conditions. Among these 24 conditions, the solution (sample value) containing human recombinant MMP-1 and the solution without human recombinant MMP are calculated. The OD value difference (sample value-background value) between -1 solutions (background value). The higher the value, the better the binding ability of the detectable MMP-1 of the antibody pair. 2. Complete 92 sets of pairing tests according to the aforementioned step 1. The results are listed in Table 1 below. The values in the table are the values with the largest difference in OD value of each pair in the pairing test.

註：差值<0.5的用「-」標示，沒有進行測試的用「NA」標示。 Table 1. Table of the maximum difference between the sample values of each pair after subtracting the background value

Note: If the difference is less than 0.5, it is marked with "-", and it is marked with "NA" if it is not tested.

Table 1 shows that the pairing of monoclonal antibodies with stronger signals (higher differences) in the pairing test has no absolute relationship with the MMP-1 binding ability of individual monoclonal antibodies. That is, the best antibody pairing suitable for the enzyme immunoassay reagent set cannot be determined by the MMP-1 binding ability of individual monoclonal antibodies, and the pairing test must be actually performed to screen.

Example II.3. Enzyme immunoassay reagent set for detecting MMP-1-standard concentration curve of each antibody pair

According to the results of Example II.2, 12 antibody pairs were selected (listed in Table 2), and the sandwich enzyme immunoassay method was performed to test the range of the human recombinant MMP-1 standard concentration curve that can be detected by individual antibody pairs. The results are presented于表3。 In Table 3.

Table 2. Comparison table of each antibody pairing

Table 3. Comparison table of standard concentration curve range for antibody pairing test

The results in Table 3 show that testing the concentration range of human recombinant MMP-1 between 0.01 and 2.5 ng/ml, the OD value of each pair increases as the concentration of MMP-1 increases.

Then, the recovery rate (Recovery) is used to evaluate the detectable standard curve concentration range of each group of antibody pairings. Recovery 80-120 is set as the reliable range, and the concentration falling within the reliable range is marked in light gray. The results showed that the concentration ranges of the standard curves of the 12 antibody pairs are different, and some pairs have negative values at low concentrations, indicating that the background value is high, which is not conducive to the detection of low concentration samples.

Example two.4. Enzyme immunoassay reagent set for detecting MMP-1-detection of endogenous MMP-1 in clinical saliva samples

Next, the 12 antibody pairs in Example 2.3 were used to detect 8 clinical saliva samples with known endogenous MMP-1 content (the content was determined by multiplex liquid chromatography-multiplex reaction monitoring mass spectrometry (Multiplex LC-MRM-MS) ) To confirm whether the antibody pairing can detect endogenous MMP-1 in clinical saliva samples in addition to human recombinant MMP-1. The results are shown in Table 4.

Table 4. Comparison of OD values of antibody pairing for detecting endogenous MMP-1 in saliva samples

The results show that all 12 antibody pairs can detect the endogenous MMP-1 in saliva samples, and the MMP-1 content measured by most of the pairs is consistent with the actual content. It shows that 12 antibody pairs are used to detect saliva samples. MMP-1 is also sufficiently specific. According to the results in Table 4, select No. 2, No. 4, No. 5, and No. 10 with better sensitivity for further evaluation.

To evaluate the feasibility of clinical application, 15 clinical saliva samples from healthy people (Healthy Control, HC) and 25 from oral cancer patients (Oral Squamous Cell Carcinoma, OSCC) were collected and diluted 5 times for enzyme immunoassay experiments. , Please see Figure 2A (normal group) and Figure 2B (oral cancer patient group) for the results. And use the 2A figure and the 2B figure for further data analysis.

The results showed that, no matter in normal people or oral cancer patient groups, the four antibody pairings measured values were significantly correlated (Spearman's rho=0.953-0.988); in addition, the receiver operating characteristic curve ( Receiver operating characteristic curve (ROC) analysis results show that all four antibody pairs are suitable for detecting endogenous MMP-1 (Area Under the Curve of ROC; AUC of ROC) in saliva samples. =0.937-0.967), the sensitivity and specificity of the four pairings obtained by ROC analysis are summarized in Table 5.

Table 5. Sensitivity and specificity analysis of antibody pairing

According to Table 5, select the antibody pair No. 4 with higher sensitivity to prepare the MMP-1 enzyme immunoassay reagent set.

Example two.5. Enzyme immunoassay reagent set for detecting MMP-1-functional test

According to the Global Laboratory Standards for a Healthier World (Global Laboratory Standards for a Healthier World) published by the American Association for Clinical and Laboratory Standards, a functional test was performed on the MMP-1 enzyme immunoassay reagent set using antibody pairing No. 4, and the results are as follows.

1. Sensitivity analysis (refer to specification Ep17-A2), blank detection The limit of blank (LoB) is 57.40 picogram/ml, and the limit of detection (LoD) of the test product is 117.02 picogram/ml.

2. Linear analysis (refer to the specification Ep06-A), the best nonlinear polynomial is a 3-level regression, and the concentration is linear distribution between 140~8000 picogram/ml (non-linearity≦5%).

3. Precision analysis (refer to the specification Ep05-A3), the average CV (%) of repeatability (repeatability) is 4.809%, and the average CV (%) of within-laboratory precision (within-laboratory precision) is 9.569%.

Example III. 1. Selection of colloidal gold immune test strip-antibody combination for detecting MMP-1

Then, further test the antibody combination of the colloidal gold immune test strip suitable for detecting MMP-1 among the self-produced monoclonal antibodies. The schematic diagram of the colloidal gold immunoassay strip 1 is shown in Fig. 3A. It includes the glue gold pad 110 and the measuring plate 120, which can be stacked on each other, for example, the glue gold pad 110 is stacked on the measuring plate 120 or the measuring plate 120 is stacked. On the glue gold pad 110, spray the glue gold pad solution 111 containing the detection antibody-gold particle coupling compound on the glue gold pad 110, the surface of the measuring plate 120 is covered with nitrocellulose membrane, and then the measuring line 122 is coated with the solution containing the capture antibody On the nitrocellulose membrane. In addition, as required, a solution containing an internal control antibody can be coated on the nitrocellulose membrane to serve as the internal control line 121. During the measurement, the sample is added to the gel gold pad 110 containing the detection antibody-gold particle conjugate, and the sample will move from the gel gold pad 110 to the nitrocellulose membrane on the measurement plate 120 through immunochromatography. MMP-1 has been bound to the detection antibody-gold particle conjugate, the capture antibody on the measuring line 122 will capture the MMP-1 bound to the detection antibody-gold particle conjugate, and then aggregate to form an orange to red signal (gold The color of the particle). Therefore, the measurement result can be judged through the color of the measurement line 122.

In order to select the combination of antibody pairings, 4 antibody pairs (No. 2, No. 4, No. 5, No. 10) that showed better sensitivity in Example II.4 were paired, and the monoclonal antibodies used were: 1. -8A12 strains, 6-2 strains, 20-4 strains, 31-34 strains and 73-1 strains, the paired combination test was performed as follows.

First, the five antibodies are reacted with nano-sized colloidal gold particles (diameter <100 nanometers) to form a "detection antibody-gold particle conjugate". However, the 1-8A12 strain and the 73-1 strain did not react well with the colloidal gold particles, and the gold particles could not be connected to the detection antibody. Therefore, only 6-2-gold particles, 20-4-gold particles, and 31-gold particles were used. 34-gold particles were paired with human recombinant MMP-1 for a total of 10 pairing combinations. The results are shown in Table 6.

Table 6. Determination limits of antibody pairing combinations used for colloidal gold immunoassay strips

According to Table 6, because combination C can achieve better sensitivity, combination C is selected for subsequent clinical sample testing.

Example III. 2. Colloidal gold immunoassay strips for the detection of MMP-1-testing of clinical samples

In order to evaluate the feasibility of using colloidal gold immunoassay strips containing combination C to detect clinical samples, 215 groups of saliva samples were tested and simultaneously used for cross-comparison with enzyme immunoassay.

For the colloidal gold immune test piece, please refer to Figure 3B. The reaction degree of the sample is scored against the colorimetric card. The colorimetric card is divided into 0~5 points according to the color intensity of the measurement line 122. If the colorimetric card is applied, the score will be 0, 0-1 (0.5), 1, 1-2 ( 1.5), 2, 2-3 (2.5), 3, 3-4 (3.5), 4, 4-5 (4.5), 5,> 5 (5.5), a total of 12 grades. Next, make an X-Y distribution graph of the grades of the colorimetric card and the MMP-1 concentration measured by the enzyme immunoassay method. The results are shown in Figure 4.

Figure 4 shows that the colloidal gold immunoassay is positively correlated with the results of enzyme immunoassay, and the correlation analysis result is R=0.871 (p<0.0001). That is, the colloidal gold immunoassay strip containing combination C can be used to detect MMP-1 in clinical saliva samples.

In summary, the embodiments of the present disclosure disclose a detection method that is effective for detecting endogenous MMP-1 in clinical samples. The detection method can at least include: enzyme immunoassay reagent, using monoclonal antibody paired to No. 4 (capture antibody: 31-34 strain and detection antibody: 73-1 strain) and colloidal gold immunoassay Use the monoclonal antibody pairing to form combination C (capture antibody: 1-8A12 strain and detection antibody: 31-34 strain). The amino acid sequence, main features, and corresponding sequence number of each monoclonal antibody are as follows 7 shown.

Table 7, 31-34, 73-1 and 1-8A12 sequence alignment table

Example four, comparison with the test results of the control group

During the development process of Examples 2 to 3, each test also uses several control groups as capture antibodies, and self-produced 1-8A12 as detection antibodies, and the test results are simultaneously compared and analyzed. The control antibodies are shown in Table 8, which includes commercially available antibodies (No. 7), MMP-1 peptide fragments as immunogens produced multiple antibodies (No. 1 to 4), and MMP-1 without protease activity as the There are 16 antibodies (numbers 5 to 6) produced by the antigen and other mouse monoclonal antibodies (numbers 8 to 16) produced using MMP-1 without protease activity as the immunogen.

Table 8. Antibody preparation process information table of the control group

The results of the comparison show that most antibodies can effectively bind to human recombinant MMP-1 at the stage of direct enzyme immunoassay. However, when entering the antibody pairing process, many pairs will have excessive non-specific binding and generally insufficient sensitivity. . In the pairing of the control group as the capture antibody and the self-produced antibody as the detection antibody, the best pairing is the commercially available antibody MAB901 with 1-8A12-HRP, but its standard concentration curve range (Table 9A) and saliva In the samples (Table 9B), the detection efficiency of MMP-1 is worse than that of self-produced antibody pairs. Therefore, the sensitivity of the pair screened in the examples of the present disclosure is better than that of capture antibodies using commercially available antibodies, other monoclonal antibodies produced using MMP-1 without protease activity as the immunogen, or using MMP-1 fragments as the immunogen. The immunogen immunizes the antibody pair to produce multiple antibodies.

Table 9A. The comparison table of the standard concentration curve range of the control group and the antibody pairing value of self-screening

Table 9B. Comparison table of the results of the control group and the self-screened antibody pairing used to detect saliva samples

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<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR1 of the light chain variable region of monoclonal antibody 31-34

<400> 4

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR2 of light chain variable region of monoclonal antibody 31-34

<400> 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR3 of light chain variable region of monoclonal antibody 31-34

<400> 6

<210> 7

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR1 of the heavy chain variable region of monoclonal antibody 73-1

<400> 7

<210> 8

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR2 of the heavy chain variable region of monoclonal antibody 73-1

<400> 8

<210> 9

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR3 of the heavy chain variable region of monoclonal antibody 73-1

<400> 9

<210> 10

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR1 of the light chain variable region of monoclonal antibody 73-1

<400> 10

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR2 of light chain variable region of monoclonal antibody 73-1

<400> 11

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR3 of the light chain variable region of monoclonal antibody 73-1

<400> 12

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR1 of the heavy chain variable region of monoclonal antibody 1-8A12

<400> 13

<210> 14

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR2 of heavy chain variable region of monoclonal antibody 1-8A12

<400> 14

<210> 15

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR3 of heavy chain variable region of monoclonal antibody 1-8A12

<400> 15

<210> 16

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR1 of light chain variable region of monoclonal antibody 1-8A12

<400> 16

<210> 17

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR2 of light chain variable region of monoclonal antibody 1-8A12

<400> 17

<210> 18

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR3 of light chain variable region of monoclonal antibody 1-8A12

<400> 18

Claims (10)

A monoclonal antibody composition, wherein the monoclonal antibody composition comprises a monoclonal antibody A and a monoclonal antibody B, or comprises a monoclonal antibody A and a monoclonal antibody C, wherein the single antibody of the monoclonal antibody A The chain variable region sequence includes the amino acid sequence of SEQ ID NO: 1, 2, and 3, and a light chain variable region sequence of the monoclonal antibody A includes the amino acid sequence of SEQ ID NO: 4, 5, and 6. Acid sequence; the heavy chain variable region sequence of the monoclonal antibody B includes the amino acid sequence of SEQ ID NO: 7, 8, and 9, and the light chain variable region sequence includes SEQ ID NO: 10, 11, And 12 amino acid sequences; and the heavy chain variable region sequence of the monoclonal antibody C includes the amino acid sequences of SEQ ID NOs: 13, 14, and 15, and the light chain variable region sequence includes SEQ ID NO: the amino acid sequence of 16, 17, and 18. A polynucleotide, wherein the polynucleotide encodes the amino acid sequence of the monoclonal antibody composition as described in claim 1, or has a nucleus that encodes the monoclonal antibody composition as described in claim 1 A sequence that is complementary to the nucleotide sequence. A test kit, wherein the test kit comprises a monoclonal antibody composition as described in claim 1. The test kit described in claim 3, wherein the test The set is an enzyme immunoassay reagent set, a colloidal gold immunoassay strip or a combination thereof. The detection kit according to claim 4, wherein the enzyme immunoassay reagent kit comprises the monoclonal antibody A and the monoclonal antibody B, and the monoclonal antibody B is connected to a chromogenic group. The detection kit according to claim 4, wherein the colloidal gold immunity test strip comprises: the monoclonal antibody A and the monoclonal antibody C. The detection kit according to claim 6, wherein the monoclonal antibody A is connected to a gold particle. An in vitro method for detecting type 1 matrix metalloproteinases, comprising: using the type 1 matrix metalloproteinases in the test kit as described in claim 3. The method according to claim 8, wherein the sample contains a body fluid or a blood. The method according to claim 9, wherein the body fluid comprises an oral secretion or a respiratory secretion.

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